Electrolyte homeostasis is essential for life at the cellular level, in that, cells must respond to osmotic challenge to fend off changes in cellular water and ion content that could lead to rupture. On a larger scale, humans regulate blood pressure by maintaining an appropriate balance of sodium reabsorption and excretion in the kidney. Hypertension, a major health problem affecting more than 60 million Americans, is a result of a dysfunction in electrolyte homeostasis. Therefore, understanding mechanisms that control electrolyte homeostasis is important for human health. It is becoming increasingly clear that a core set of regulatory proteins senses and maintains electrolyte homeostasis. Our knowledge is lacking in how this is achieved at the molecular level. The molecular mechanisms of electrolyte homeostasis are of critical importance for both healthy and disease states in humans and thus must be understood in order unlock their therapeutic potential. We aim to understand the network of proteins and signaling mechanisms, mainly regulatory phosphorylation events, which connect mechanisms of cell volume control and blood pressure homeostasis. The red blood cell holds great potential as a model system to understand the fundamental elements critical for electrolyte homeostasis. We will use a quantitative proteomic approach to study networks of signaling proteins that regulate electrolyte flux in red blood cells. We will focus our studies on the K-Cl cotransporters as a representative direct mediator of electrolyte flux and the kinases Wnk1 and Wnk4 as critical signaling components of ion flux. These studies will provide new insight into the upstream regulation of Wnk function and downstream signaling events that control electrolyte homeostasis by identifying critical regulatory phosphorylation sites. To link these observations to the in vivo setting, we will use SILAC technology in the mouse red blood cell to quantify critical regulatory phosphorylation sites that respond to specific physiologic perturbation. The purpose of this study is to provide a fundamental understanding of the mechanisms that coordinate electrolyte homeostasis. Furthermore, we are seeking mechanistic links in blood pressure control and cell volume regulation in order to find new target points to treat diseases such as hypertension and sickle cell anemia.